Project summary Streptococcus pneumoniae is a Gram-positive respiratory pathogen responsible for life-threatening infections in young children and older adults. Patients with pneumococcal infections are typically treated with antibiotics that act by targeting the assembly of the cell wall. Over the last decade S. pneumoniae has become a serious health threat with multidrug resistance increasing at alarming rates. There is therefore an urgent need to develop new and effective therapies. Given that the cell envelope is the primary target of many of our best treatments, studies aimed at understanding the mechanisms controlling its assembly hold promise in defining new vulnerabilities that can be blocked for the treatment of disease. The proposed research will address three fundamental areas of S. pneumoniae cell envelope assembly and morphogenesis. The first is the regulation of the major autolysin LytA, an enzyme that cleaves the cell wall peptidoglycan (PG) matrix and is responsible for cell lysis following entry into stationary phase or upon treatment with beta-lactam antibiotics. LytA activity must be tightly regulated to prevent cell wall damage that can lead to lysis. However, the mechanism by which this enzyme is controlled has remained mysterious. It is also unclear how this control is subverted by beta-lactam antibiotics and other anti-cell wall drugs to elicit lysis. The second area involves the biochemical pathways that build the major surface polymers called teichoic acids (TAs), which are critical for host colonization and virulence. My preliminary results have revealed an exciting connection between LytA regulation and TA biogenesis, providing an opportunity to uncover mechanistic insight into both areas. I discovered that a novel membrane protein (RafX) appears to be involved in the biosynthesis of TAs, and in blocking LytA-dependent autolysis. Finally, the third area relates to how the division site is specified in S. pneumoniae. Currently, the only known factor involved in division placement in this bacterium is not essential and its inactivation results in mild division placement defects. Other regulators are therefore predicted to exist, but remain to be identified. This aim will be facilitated by a screen for mutants synthetically lethal with known regulator that I have carried out. The Specific Aims of this F32 application are: 1) Define the mechanism by which RafX modulates TA assembly and how this activity controls LytA autolysis. 2) Determine the mechanism of division site placement in S. pneumoniae.